Genetic identification of SNP markers linked to a new grape phylloxera resistant locus in <Emphasis Type="Italic">Vitis cinerea</Emphasis> for marker-assisted selection

Background

Grape phylloxera (Daktulosphaira vitifoliae Fitch) is a major insect pest that negatively impacts commercial grapevine performance worldwide. Consequently, the use of phylloxera resistant rootstocks is an essential component of vineyard management. However, the majority of commercially available rootstocks used in viticulture production provide limited levels of grape phylloxera resistance, in part due to the adaptation of phylloxera biotypes to different Vitis species. Therefore, there is pressing need to develop new rootstocks better adapted to specific grape growing regions with complete resistance to grape phylloxera biotypes.

Results

Grapevine rootstock breeding material, including an accession of Vitis cinerea and V. aestivalis, DRX55 ([M. rotundifolia x V. vinifera] x open pollinated) and MS27-31 (M. rotundifolia specific hybrid), provided complete resistance to grape phylloxera in potted plant assays. To map the genetic factor(s) of grape phylloxera resistance, a F₁ V. cinerea x V. vinifera Riesling population was screened for resistance. Heritability analysis indicates that the V. cinerea accession contained a single allele referred as RESISTANCE TO DAKTULOSPHAIRA VITIFOLIAE 2 (RDV2) that confers grape phylloxera resistance. Using genetic maps constructed with pseudo-testcross markers for V. cinerea and Riesling, a single phylloxera resistance locus was identified in V. cinerea. After validating SNPs at the RDV2 locus, interval and linkage mapping showed that grape phylloxera resistance mapped to linkage group 14 at position 16.7 cM.

Conclusion

The mapping of RDV2 and the validation of markers linked to grape phylloxera resistance provides the basis to breed new rootstocks via marker-assisted selection that improve vineyard performance.

     

(A)sexual reproduction – a review of life cycles of grape phylloxera, Daktulosphaira vitifoliae

Since its emergence as a pest of grapevine about 150 years ago, studies on the life cycle and mode of reproduction of grape phylloxera ( Daktulosphaira vitifoliae Fitch) (Homoptera: Phylloxeridae) have been of considerable scientific interest and importance for applied viticulture. The life cycle of grape phylloxera, as described in the mid to late nineteenth century, is still subject to discussion and confusion. In this article, we aim to provide for the first time an overview of the reported life cycle strategies of grape phylloxera by summarizing documented observations, the results of controlled field and laboratory assays, and the results of molecular genetic analyses. Thus, we present a detailed morphological comparison of grape phylloxera morphs and provide clarification of these by suggesting a simplified nomenclature as well as revising errors found in the published literature. Moreover, recent molecular markers studies, which further enhance our understanding of the impact of sexual recombination on grape phylloxera, are described and discussed.     

RAPD assessment of California phylloxera diversity

Grape phylloxera, Daktulosphaira vitifoliae Fitch, is a parthenogenetic, aphid-like pest which attacks the roots and leaves of many Vitis L. species. Feeding on the roots of V. vinifera L. cultivars leads to decreased plant productivity and eventual death. Isolates of phylloxera have been grouped into ‘biotypes’ based on the aggressiveness of their feeding behaviour, relative reproductive rates, and abilities to cause decline on V. vinifera and the grape rootstock AXR1. The goal of our research was to determine whether these bio-type definitions were reflected in the genetic diversity among laboratory-cultured colonies of Californian phylloxera. Thirteen phylloxera isolates, encompassing biotypes A, B and non-A or -B types based on their feeding and reproduction, were analysed using the RAPD-PCR method. Results indicate that there were genetic differences among isolates and that there was as much polymorphism within biotypes as among biotypes. This is surprising in a parthenogenetic organism and leads us to suggest that there have been multiple introductions of phylloxera into California, that phylloxera are not obligately parthenogenetic, or that there are other mechanisms whereby phylloxera may evolve relatively rapidly.     

葡萄花翅小卷蛾抗药性研究进展

葡萄花翅小卷蛾是葡萄上的重要害虫,具有多食性、多化性等生物学特点,抗逆能力极强。该虫起源于欧洲,现已入侵全球多个国家。葡萄花翅小卷蛾主要以幼虫取食葡萄花序、幼果和成熟果实,给葡萄生产造成重大损失;其危害有利于真菌的侵入,导致灰霉病、白粉病等病害大量发生,从而造成葡萄腐烂。由于该虫入侵风险极高,已被我国列为重要的进境检疫性有害生物。国外对葡萄花翅小卷蛾的防治主要采用化学杀虫剂,由于长期大量且不合理地使用化学杀虫剂,葡萄花翅小卷蛾已对多种不同类型的杀虫剂产生了抗药性。本研究总结了葡萄花翅小卷蛾的抗性测定方法、抗性现状及其抗性机理,同时结合国外葡萄花翅小卷蛾抗性和防治相关研究,提出该虫抗性治理策略,并对我国预防该虫的入侵提出建议。     

部分葡萄品种和砧木抗葡萄根瘤蚜性能鉴定

为了检测我国葡萄品种和砧木对中国生态型葡萄根瘤蚜Daktulosphaira viticola (Fitch) 的抗性,在实验室条件下采用离体根接种法鉴定了10个葡萄砧木、18个葡萄栽培品种,测定了根瘤蚜存活率、发育历期、产卵量、种群龄期结构指数和成虫大小等指标。结果表明： 砧木品种SO4,5BB,1103P,420A,Lot,101-14MG,3309C,140Ru和110R抗根瘤蚜,接种后蚜虫陆续死亡,尤其是SO4,5BB,1103P和420A对根瘤蚜高抗,使根瘤蚜滞育仅发育至1龄。我国当作砧木使用的贝达不抗根瘤蚜,接种后蚜虫存活率始终保持在38.82％～47.5%,日产卵量达9.01;欧美杂交种中的康克、康拜尔和卡它巴对根瘤蚜稍有抗性,根瘤蚜日产卵量在6.00～6.85粒之间,其中康克、康拜尔葡萄上蚜虫的存活率随时间的延长而明显降低,由最初的60％下降至25％～30％; 欧美杂交种中的黑虎香和白香蕉虽然在一定程度上能够限制根瘤蚜的存活和发育进程,但对根瘤蚜的繁殖量没有影响。所有供试欧亚种和欧美杂交种的栽培品种都对根瘤蚜高度敏感,接种后第29 d 蚜虫存活率仍保持在40％～60％,且前后变化较小,日产卵量变化于7～12.35粒之间,总产卵量均在190粒以上。由此可见,我国供试栽培品种均不抗根瘤蚜,在根瘤蚜疫区需要用抗性砧木嫁接苗建园。     

世界性害虫葡萄花翅小卷蛾入侵我国的风险分析

【目的】葡萄花翅小卷蛾是我国禁止进境的检疫性害虫,一旦入侵我国,将会给葡萄产业和林果业造成严重损失。开展葡萄花翅小卷蛾入侵我国的风险分析可为该虫的检疫和预警提供依据。【方法】介绍了葡萄花翅小卷蛾的形态特征和生物学特性,并参照通用的有害生物风险评估方法,从该虫在国内外的发生情况、潜在的经济危害性、受害作物的经济重要性、传播扩散的可能性及风险管理的难度等方面,对其入侵风险进行了定性和定量分析。【结果】葡萄花翅小卷蛾是一种对我国有很大潜在威胁的有害生物,其风险值为2.14,属高度危险性有害生物。【结论】建议新疆等口岸比较多的省、自治区口岸检疫部门加强对该害虫的检疫力度,杜绝其传入我国。     

The Sublethal Effects of β-Ecdysterone,a Highly Active Compound from Achyranthes bidentata Blume,on Grape Phylloxera,Daktulosphaira vitifoliae Fitch

Grape phylloxera, Daktulosphaira vitifoliae (Fitch) (Hemiptera, Phylloxeridae), is a very destructive insect pest of grapevines. Intercropping of Achyranthes bidentata Blume (f. Amaranthaceae) and Vitis spp. grapevines can be useful to control this pest. In the present study, the toxicity of 22 compounds, known to be present in A. bidentata, to grape phylloxera was evaluated. All treatments were toxic towards grape phylloxera but the degree of toxicity differed between treatments. Among the 22 tested compounds, several of which proved toxic towards grape phylloxera. However β-ecdysterone had higher toxic effects against grape phylloxera, with LC₅₀ values of 175.73 mg a.i. liter^-1. In addition, we assessed the sublethal effects of LC₁₀, LC₂₀ and LC₄₀ of β-ecdysterone on grape phylloxera. The fourth instar and adult developmental periods and total life span were significantly prolonged by LC₄₀ of β-ecdysterone. Fecundity decreased when grape phylloxera were exposed to LC₂₀ and LC₄₀ of β-ecdysterone. In addition, LC₄₀ of β-ecdysterone decreased the intrinsic rate of increase (r_m) and the finite rate of increase (λ) and prolonged the population doubling time (DT). The net reproductive rate (R₀) was significantly reduced by both the LC₂₀ and LC₄₀ β-ecdysterone treatments. Our results demonstrated that β-ecdysterone had higher toxic effects and significant sublethal effects on grape phylloxera, and showed potential control of grape phylloxera.     

Heritability of adventitious rooting of grapevine dormant canes

One of the most important viticultural characteristics of a grapevine rootstock is the ability to form roots on dormant lignified canes (rootstrike). North American species of Vitis are the primary source of germplasm for grapevine rootstocks and vary widely in their rate of rootstrike. Breeders have hybridized grape species in order to introgress traits to produce commercial rootstocks. A combination of 26 parents consisting of improved and wild accessions of Vitis spp. was used to generate 27 families. The percentage of rootstrike of dormant canes was observed over several years for 552 individuals. A logistic generalized linear mixed model (GLMM) method was used to estimate the narrow sense heritability (h ²) of rootstrike. Heritability was found to be moderate (h ²?=?0.307?±?0.050). The model also estimated breeding values of all parents and progeny. A GLMM method can be used to estimate breeding values of germplasm to identify individuals with commercially acceptable rates of rootstrike with a defined probability of transmitting this trait to progeny. This is useful for the introgression of traits into potentially new commercial rootstocks. The pattern of normal distribution of rooting indicates that it is possible to identify individuals with good rootstrike from Vitis species that are generally considered to have low rootstrike. Selection of individuals with a higher breeding value will increase the efficiency of rootstock breeding.     

Evaluating resistance of some rootstocks to grape phylloxera with in vitro and excised root testing systems

Summary The life table of a local strain of grape phylloxera was determined to evaluate the resistance of the most commonly used rootstocks: Rul40, R99, and 3309C, and one local variety ‘Helwani’. The study was carried out by applying both in vitro dual culture and small root pieces testing systems. The results showed that there was a great variation in percentage mortality of immature stages, number of eggs laid, adult fertility, oviposition period, and developmental time between all tested rootstocks and the local variety, regardless of the applied testing systems. Based on the population prediction, Helwani would be a suitable host as it would be susceptible to the destructive insects in the field and the rootstocks would be resistant. However, Rul 40 rootstock was more susceptible than R99 and 3309C rootstocks.     

根瘤蚜对不同抗性葡萄的选择性及葡萄根系挥发性物质的鉴定

为了探讨不同抗性水平砧木及品种对根瘤蚜吸引及驱避效应以及鉴定挥发性物质组分,采用离体根段培养法测定葡萄根瘤蚜Phylloxera viticola Fitch对砧木5BB,140Ru及栽培品种巨峰的选择性,发现根瘤蚜对栽培品种巨峰的根段具有很强的选择性,至接种后3 d,67.13％的根瘤蚜优先选择巨峰;而对5BB和140Ru的选择性却很差,分别有15.29％和17.58％的根瘤蚜选择5BB及140Ru。根瘤蚜在不同品种根段上的生长发育和繁殖也存在明显差异。对砧木5BB和巨峰根系的挥发性组分进行测定,发现2个品种挥发物独有组分数量均较高,巨峰独有组分为23个,占总成分数的48.94％,5BB的独有组分为32个,占总成分数的57.14％。5BB和巨峰的共有组分为24个,主要成分均为亚油酸甲酯和反油酸甲酯,但含量有所不同,其中,5BB以倍半萜类的丁香醇及脂肪酸甲酯类的亚油酸甲酯,反油酸甲酯,棕榈油酸甲酯含量较高,高于巨峰中相应物质含量的4.23%~6.46％,而巨峰以邻苯二甲酸二丁酯及胆甾烷含量较高,分别高于5BB相应组分含量的6.81％和1.07％。结果说明葡萄根瘤蚜对不同抗性水平砧木及品种确实存在不同的选择性,并且不同抗性水平的砧木及品种的挥发性物质存在显著差异。     

Variation in performance on two grape cultivars within and among populations of grape Phylloxera from wild and cultivated habitats

When an indigenous insect becomes a pest, comparisons of performance of pest and non-pest populations on crop plants and of genetic variation in that performance may provide insight into the evolution of pest populations. To measure such genetic variation, 8–15 clones of the grape phylloxera (Daktulosphaira vitifoliae Fitch) were collected from wild grapevines in each of 3 geographically isolated sites (populations) and from commercial vineyards in northern California. A complete life table was made for clonal replicates from populations collected from wild grapevines on each of two commercial grape cultivars, the susceptibleVitis vinifera (L.) cultivar Cabernet Sauvignon, and the phylloxera-resistant rootstock ‘AxR # 1’. Variation in mean performance on these two hosts was partitioned among clones within collection sites and among sites. Performance measures included an individual analog to the intrinsic rate of increase (r), age at first oviposition, fecundity in the first ten days of reproduction, total fecundity, and longevity. The overall performance of phylloxera from the wild grapevines on the resistant cultivar AxR # 1 was greater than or equal to that on the susceptible cultivar Cabernet Sauvignon. There was significant variation among clones within populations from wild grapes in the rate of increase on ‘AxR # 1’ and marginally significant clonal variation in some of the component paramters. There was no significant variation among clones within populations on ‘Cabernet Sauvignon’ and no significant differences between populations on either crop in any trait. In a second experiment we compared the relative performance of 15–17 clones from wild grapevines and from commercial vineyards when reared on ‘Cabernet Sauvignon’ and ‘AxR # 1’. Phylloxera from commercial vineyards had much higher overall performance on ‘Cabernet Sauvignon’ than did phylloxera from the wild grapevines. Phylloxera from the commercial vineyard also had higher performance on ‘Cabernet Sauvignon’ than on ‘AxR′ 1’ but the performance of the phylloxera from wild and commercial grapes did not differ on ‘AxR # 1’. Our results show that there is genetic variation in traits related to performance on a resistant rootstock within these indigenous non-pest populations of phylloxera, but not among them. The pattern of performance of pest and non-pest populations on two commercial cultivars suggests that current levels of phylloxera performance on crop cultivars are the result of adaptation to those cultivars which has occurred while phylloxera has been associated with viticulture. Implications of these results for understanding the recent adaptation of phylloxera to ‘AxR # 1’ in California are also discussed.     

Performance of native grape phylloxera on host plants within and among terrestrial islands in Arizona, USA

In Arizona, USA, the canyon grape, Vitis arizonica Englemann, and grape phylloxera (Daktulosphaira vitifoliae Fitch, Homoptera, Phylloxeridae) are distributed among mountain ranges that are surrounded by expanses of desert lacking Vitis habitat, thus forming a system of terrestrial islands. Both herbivore and host populations may have diverged genetically among mountain ranges under the influence of restricted gene flow and variable selection among sites. Herbivore adaptation to local hosts would be expected to ensue, with the potential to promote divergence, both in traits under selection and by further reducing the probability of interisland colonization. To test the hypothesis that phylloxera are adapted to local hosts, demographic components of fitness of field-collected native grape phylloxera were measured in the greenhouse on vines of V. arizonica that were categorized as either natal, neighboring, and or isolated hosts. There was no evidence for greater adaptation to natal or neighboring hosts but there were significant interactions between herbivore and host treatments in one experiment. There was genetic variation for gall formation among six clones tested. Though a failure to detect local adaptation could have resulted from low statistical power, benign experimental conditions, or inadequate genetic variation, the divergence of isolated grape populations is suggested to have been insufficient to promote local adaptation in grape phylloxera at the level of isolated mountain ranges. It is further suggested that, within populations, adaptation to individual host plants could be forestalled by selection for ’general purpose genotypes’ through wind-borne displacement of colonizers into the unpredictable environment of a heterogeneous array of hosts. In addition, short-term extinction/colonization dynamics could promote gene flow such that time is insufficient for adaptive mutations or gene combination to arise. Received: 26 December 1998 / Accepted: 24 May 1999     

Clonal reproduction and population genetic structure of grape phylloxera, Daktulosphaira vitifoliae, in Australia

The grape phylloxera, Daktulosphaira vitifoliae, is a viticultural pest that in the past has devastated vineyards worldwide, yet little is known about this insect's biology. The genetic structure of Australian populations of grape phylloxera and its mode of reproduction were studied following the development of four polymorphic microsatellite loci. Insects were collected from 28 vineyards, with a total of 361 insects included in the study. The majority of vineyards were infested by functionally parthenogenetic lineages of grape phylloxera that inhabit the root system and there was little support for the traditionally described holocyclic life cycle for this species. Clonal diversity was limited in all of the vineyard regions, with the exception of the Rutherglen region. A multiple founder scenario or occasional sex may contribute to diversity within the Rutherglen region. Leaf galling populations comprised classes distinct from the common genotypic classes identified on the roots, suggesting limited exchange between these groups. Implications for the management of D. vitifoliae are discussed.     

Landscape simplification increases vineyard pest outbreaks and insecticide use

Diversifying agricultural landscapes may mitigate biodiversity declines and improve pest management. Yet landscapes are rarely managed to suppress pests, in part because researchers seldom measure key variables related to pest outbreaks and insecticides that drive management decisions. We used a 13‐year government database to analyse landscape effects on European grapevine moth (Lobesia botrana) outbreaks and insecticides across c. 400 Spanish vineyards. At harvest, we found pest outbreaks increased four‐fold in simplified, vineyard‐dominated landscapes compared to complex landscapes in which vineyards are surrounded by semi‐natural habitats. Similarly, insecticide applications doubled in vineyard‐dominated landscapes but declined in vineyards surrounded by shrubland. Importantly, pest population stochasticity would have masked these large effects if numbers of study sites and years were reduced to typical levels in landscape pest‐control studies. Our results suggest increasing landscape complexity may mitigate pest populations and insecticide applications. Habitat conservation represents an economically and environmentally sound approach for achieving sustainable grape production.     

Progress in grapevine breeding

Summary The European, or bunch grape, Vitis vinifera, is widely grown because of its high fruit quality and its capacity to grow in a wide range of climatic conditions. However, they are susceptible to fungal diseases and insect pests, especially when grown in cool, wet climates. The aim of a number of grapevine breeding programs throughout the world is to develop new varieties resistant to diseases using complex hybrids between European and American species of Vitis. Within these breeding programs it is essential to maintain heterozygosity and desirable hybrids are multiplied by asexual propagation. New approaches to grapevine improvement include the use of protoplast fusion to overcome sexual barriers, however the routine regeneration of plantlets from protoplasts and calluses is difficult. In vitro rescue of ovules from varieties with stenospermocarpic seeds shows considerable promise for breeding new seedless grapes. Eventually the use of plant transformation techniques to insert specific pieces of DNA coding for desirable genetic characteristics will provide opportunities for equipping well known grape cultivars with new characteristics.     

An assessment method for the quantification of Daktulosphaira vitifoliae (Fitch) (Hem., Phylloxeridae) populations in the field

Abstract:  Grape phylloxera is a plant parasite that has started to become a pest again in European viticulture in spite of the use of grafted rootstocks. Since the middle of the 1980s, there have been various reports of vine damage caused by phylloxera. It was shown that in some cases there is no direct correlation between the occurrence of phylloxera and vine damage. In this context, phytopathogenic microorganisms, especially fungi, are mentioned in literature. To investigate the possible interactions it is necessary to be able to assess phylloxera populations in the field, e.g. to correlate phylloxera abundance with the vigour of the vines. Because of the non-homogenous distribution of the grape roots in the soil and of phylloxera on the roots, a method that meets these conditions was established. For the root sampling, a method that ensures reproducible numbers and a high probability of root detection was developed. The second component of the method is a ranking system for the visual assessment of the populations. For the validation of the assessment system, the absolute number of phylloxera feeding sites, all phylloxera instars and phylloxera eggs were related to the weight of fresh and dry roots. The statistical analysis has shown a significant differentiation between the assessment classes (P ≤ 0.01). Furthermore, a correlation ( R ² = 0.732) between the phylloxera populations, assessed with this method, and soil temperatures could be shown. Such a correlation was shown in various studies and, therefore, supports the validity of the assessment system presented in this paper.     

Changes in grape phylloxera abundance in ungrafted vineyards

To examine seasonal changes in the abundance of grape phylloxera Daktulosphaira vitifoliae (Fitch), several sampling methods were tested at vineyards in Victoria, Australia. At a recently infested site, changes detected by root assessment, trunk trapping, and emergence trapping were closely correlated, although the largest numbers of grape phylloxera were obtained using traps that collected phylloxera emerging from soil. This trapping technique was further used to investigate changes in grape phylloxera numbers across three different sites from southeastern Australia as well as in three consecutive seasons at the same vineyard. Grape phylloxera numbers decreased as vines deteriorated; a single peak of emergence occurred in every summer. Size and timing of emergence peaks varied between sites and also between vine blocks within a site. The number of grape phylloxera trapped was correlated with degree-days. Monitoring soil temperature may provide a way of timing control options against grape phylloxera and a way of identifying peak periods when phylloxera detection surveys should be completed or when grape phylloxera are at the highest risk of spreading among vineyards.     

Relative performance of European grapevine moth (<Emphasis Type="Italic">Lobesia botrana</Emphasis>) on grapes and other hosts

The European grapevine moth, Lobesia botrana is a major grapevine pest, but despite the abundance of vineyards it is a generalist and uses either grapes or alternative species. Given the abundance and predictability of grape, L. botrana could be expected to have evolved towards monophagy. In order to understand why this species remains polyphagous, we hypothesized that larvae reared on rare wild host plants should have higher fitness than those reared on the more abundant grape host. For this, we compared larval performance and several life history traits on three alternative host plants (Daphne gnidium, Olea europaea, Tanacetum vulgare) and three Vitaceae (Vitis vinifera), two cultivars and one wild species (Ampelopsis brevipedunculata), and two control groups raised on either a low or a high nutritive value medium. Alternative hosts are more suitable than Vitaceae for the reproductive performance of L. botrana: larval mortality and development time was reduced, while pupal weight, growth rate, female longevity, female fecundity, duration of laying and mating success were increased. High quality food ingested by larvae promotes higher adult body weight and enhances female reproductive output. This suggests that alternative hosts provide greater nutritional value for L. botrana than Vitaceae. The use of alternative host plants could thus be maintained in the host range because they offer L. botrana a better fitness than on the Vitaceae. This could typically represent an advantage for moths behaving in plant diversity grape landscapes.     

Cryptic diversity of <Emphasis Type="Italic">Plasmopara viticola</Emphasis> (Oomycota,Peronosporaceae) in North America

Plasmopara viticola is the causal agent of grapevine downy mildew and is among the most important diseases in viticulture. It originates from North America, where it coevolved with wild Vitis species. Beginning in the 1870s it turned into a global epidemic that has been causing severe yield losses. It is generally believed that a single species is causing downy mildew on a large variety of economically important cultivars. Here we report, based on one nuclear and two mitochondrial markers, that isolates from vineyards in the United States fall into three highly distinct phylogenetic lineages. One of these contains European strains and affects Vitis vinifera cultivars, while the other two lineages affect also other species of Vitis. The divergence between these lineages is high, and, judging from the genetic variation in other Plasmopara lineages, might reflect distinct species. Due to the potentially significant implications for quarantine regulations and resistance breeding, detailed studies will be necessary to clarify whether these genetically distinct lineages occur outside of North America or are still confined there.